Optical imaging
Spectroscopic
applications
Microscopes
conventional optical microscope confocal microscop
measurement of fluorescence lifetime Endoscopes, telescopes
Photomultipliers, microchannelplate detector gated image intesifiers
Streak camera
CCD and CMOS cameras
Bazsalikom levél,
szekréciós mirigy az epidermisz felszínén Fluorescence from a basil leaf
(cell walls fluoresce in the blue-green region, red fluorescence is due to chlorophyll)
exc= 351 nm
em= 385-470 nm blue 515-550 nm green > 650 nm red
100 m
Sorghum
Sorghum levél felszine: levél felszine:
excexc= 351 nm= 351 nm
emiemi= 385-470 nm kék= 385-470 nm kék
515-550 nm zöld515-550 nm zöld
> 650 nm vörös> 650 nm vörös
A
100 m
A B
D C
B D
C
32 m 32 m
Fluorescence layer by layer from a sorghum leaf
On the surface the blue-green fluorescence of cell walls dominates, the chloroplasts lie deeper
exc = 351, 364nm, Ar laser
505 nm <em <550 nm
em > 650 nm
DanePy infiltrated spinach leaf, at a depth of 15 m
Distance (m)
0 20 40 60 80 100 120
Fluorescence intensity (a.u.)
0 50 100 150 200 250
t=0, > 650 nm
t=0, 505 nm < < 550 nm
Composite picture (green + red fluorescence) and intensity distribution along the line
exc = 351, 364nm
505 nm <em <550 nm
em > 650 nm
DanePy infiltrated spinach leaf
After 45 minutes photoinhibition treatment:
• 2/3 of the photosynthetic activity has been lost,
• only 15% protein degradation can be measured
• no significant pigment bleach (total pigment) or lipid peroxidation
0’ 15’ 30’ 45’
Principle of multiphoton excitation
Scales in Microscopy
Conventional light microscopy is limited by diffraction
Approaches in super-resolution light microscopy
Schermelleh L, Heintzmann R, . et.al.
STED (Stimulated Emission Depletion Microscopy)
SIM (Structured Illumination Microscopy)
SINGLE-MOLECULE IMAGING (STORM/PALM)
Instrumentation
N-STORM implementation by nikon
Fluorescence lifetime
imaging
Measurement of temperature
based on
phosphorescenc lifetime
Fotoelektronsokszorozók
Fotoelektronsokszorozó működési elve
Microchannel plate detector működési elve
Kapuzható képerősítő működési elve
Példa a kapuzott képerősítő használatára
Streak camera működési elve
Streak camera használata fotokróm spirobenzopirán vizsgálatában
Egy felvétel
Typical Quantum Efficiency Curves
0 10 20 30 40 50 60 70 80 90 100
200 300 400 500 600 700 800 900 1000
Wavelength (nm)
Back-illuminated
Front-illuminated MicroLens
front-illuminated Gen III+
Quantum Efficiency (%) ‘Virtual Phase’
Front-illuminated
Interline CCD
No Gain EM Gain
No Gain EM Gain
BODIPY
Texas Red
EMCCD enhancing conventional epi-fluorescence microscopy?
